Hello all! im doing cytotoxicity assays using PKH26 as a membrane stain measuring decrease of fluorescence intensity by flow cytometry, is it possible to use the passbands 525/50 and 530/30 in a LSR Fortessa Cytometer having in mind that exitation of this compunt is 551nm and emission of 567nm using a 488nm exitation laser
You can use the 488nm laser but ideally you should use the 575/26 or 585/42 filter. The main problem is that with the 488nm laser you won't get the maximum fluorescence but you will still be able to detect it. If those filters are the only ones available I believe the 525/50 might work better.
Take a look at the paper I've attached, they show flow cytometry analysis using PKH26.
http://cancerres.aacrjournals.org/content/53/10/2358.full.pdf+html
What about using another laser source, i got 405nm and 561nm!!! thanks in advance for the quick replay
You can use the 488nm laser that won't be a problem as long as you don't use any FITC or Alexa488 antibody with it. Are you using any viability stain with it (PI or 7AAD)?
The JOVE protocol below is very well detailed. They even use LSRFortessa.
http://www.jove.com/video/4287/optimized-staining-proliferation-modeling-methods-for-cell-division
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